Renin is an endopeptidase (molecular weight about 40,000) produced and secreted by the juxtaglomerular cells of the kidney, which cleaves its plasma substrate, angiotensinogen, specifically at the 10, 11 peptide bond, i.e., between Leu 10 and Leu 11 in the equine substrate, as described by Skeggs et al., J. Exper. Med. 1957. 106, 439, or between the Leu 10 and Val 11 in the human renin substrate, as elucidated by Tewksbury et al., Circulation 59, 60, Supp. II: 132, Oct. 1979. Renin cleaves angiotensinogen to split off the decapeptide, angiotensin I, which is converted by angiotensin-converting enzyme to the potent pressor substance angiotensin II. Thus, the renin-angiotensin system plays an important role in normal cardiovascular homeostasis and in some forms of hypertension.
Inhibitors of angiotensin I converting enzyme have proven useful in the modulation of the renin-angiotensin system and consequently, specific inhibitors of the limiting enzymatic step that ultimately regulates angiotensin II production, the action of renin on its substrate, have also been sought as effective investigative tools and as therapeutic agents in the treatment of hypertension and congestive heart failure.
Renin antibody, pepstatin, phospholipids, and substrate analogs, including tetrapeptides and octa- to tridecapeptides, with inhibition constants (K.sub.i) in the 10.sup.-3 to 10.sup.-6 M region, have been studied.
Umezawa et al., in J. Antibiot. (Tokyo) 23: 259-262, 1970, reported the isolation of a peptide, pepstatin, from actinomyces that was an inhibitor of aspartyl proteases such as pepsin, cathepsin D, and renin. Gross et al., Science 175:656, 1972, reported that pepstatin reduces blood pressure in vivo after the injection of hog renin into nephrectomized rats, but pepstatin has not found very wide application as an experimental agent because of its limited solubility and its inhibition of a variety of other acid proteases in addition to renin.
Many efforts have been made to prepare a specific renin inhibitor based on pig renin substrate analogy, which as been shown to correlate well with and predict human renin inhibitor activity. The octapeptide amino acid sequence extending from histidine-6 through tyrosine-13 ##STR1## has been shown to have kinetic parameters essentially the same as those of the full tetradecapeptide renin substrate.
Kokubu et al., Biochem. Pharmacol., 22, 3217-3223, 1973, synthesized a number of analogs of the tetrapeptide found between residues 10 to 13, but while inhibition could be shown, inhibitory constants were only of the order of 10.sup.-3 M. Analogs of a larger segment of renin substrate were synthesized, Burton et al., Biochemistry 14: 3892-3898, 1975, and Poulsen et al., Biochemistry 12: 3877-3882, 1973, but a lack of solubility and weak binding (large inhibitory constant) have proven to be major obstacles to obtaining effective renin inhibitors.
Modifications to increase solubility soon established that the inhibitory properties of the peptides are markedly dependent on the hydrophobicity of various amino acid residues, and that increasing solubility by replacing lipophilic amino acids with hydrophilic isosteric residues becomes counterproductive. Other approaches to increasing solubility have had limited success.
Modifications designed to increase binding to renin have also been made, but here too, with mixed results.
Powers et al., in Acid Proteases, Structure, Function and Biology, Plenum Press, 1977, 141-157, have suggested that in pepstatin, statine occupies the space of the two amino acids on either side of the cleavage site of a pepsin substrate, and Tang et al., in Trends in Biochem. Sci., 1:205-208 (1976) and J. Biol. Chem., 251:7088-94, 1976, have proposed that the statine residue of pepstatin resembles the transition state for pepsin hydrolysis of peptide bonds. Inhibitors of renin which contain the amino acid statine have been disclosed in the following: Veber et al, U.S. Pat. No. 4,384,994; European Published Application No. 77 029; Evans et al, U.S. Pat. No. 4,397,786; Veber et al, EP-A No. 77 028; Boger et al, Nature, 1983, 303, 81-84; U.S. Pat. No. 4,470,971; EP-A No. 114 993 and No. 157 409; U.S. Pat. No. 4,485,099; Matsueda et al, EP-A No. 128 762, 152 255; Morisawa et al., EP-A No. 186 977; Riniker et al, EP-A No. 11 266; Bindra et al, EP-A No. 55 809; Stein et al, Fed. Proc. 1986, 45, 869; and German Patent Application DE No. 3438-545-A.
Renin inhibitors containing other peptide bond isosteres, including a reduced carbonyl peptide bond isostere are disclosed by M. Szelke et al, in work described in published European Patent Application Nos. 45 665, 104 041, U.S. Pat. No. 4,424,207, and in PCT Int. Appl. WO No. 84 03,044; Nature, 299, 555 (1982); Hypertension, 4, Supp. 2, 59, 1981; British Pat. No. 587,809; and in Peptides, Structure and Function: Proceedings of the Eighth American Peptide Symposium, ed. V. J. Hruby and D. H. Rich, p. 579, Pierce Chemical Co., Rockford, Ill., 1983, where substitution at the Leu-Leu site of renin cleavage by isosteric substitution, resulted in compounds with excellent potency. Other peptide bond isosteres have been disclosed in Buhlmayer et al in EP-A No. 144 290 and No. 184 550; Hester et al. EP-A No. 173 481; Raddatz, EP-A No. 161 588; Dann et al, Biochem. Biophys. Res. Commun. 1986, 134, 71-77; Fuhrer et al., EP-A No. 143 746; Kamijo et al, EP-A No. 181 110; Thaisrivongs et al. J. Med. Chem., 1985, 28, 1553-1555; Ryono et al., EP-A No. 181 071; and Evans et al, U.S. Pat. No. 4,609,641.
Of these non-statine peptide bond isosteres, (3S, 4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA, an analog of statine in which the isobutyl group has been replaced by a cyclohexylmethyl group), when substituted for statine in a renin inhibitor, has often resulted in inhibitors that are 50-fold more potent than the corresponding statine-containing analogs. Previous methods for synthesizing the necessary protected derivative of enantiomerically-pure ACHPA for producing these useful renin inhibitors, however, have been limiting. See, e.g., Boger et al., J. Med. Chem. 1985, 28, 1779-1790, wherein low yields, difficulties in scale-up and the ease of racemization of the reductively-derived alpha-amino aldehydes produced significantly impeded development of potent, orally-active inhibitors of renin.
Descamps et al., in EPO published application No. 165 226 (1985), described a synthesis of (3S, 4S)-(N.alpha.-BOC-4-amino-5-cyclohexyl-3-hydroxypentanoic acid methyl ester, in which the magnesium salt of malonic acid monomethyl ester is used to prepare a keto ester intermediate, which is subsequently reduced to yield the product as a mixture of (3S, 4S) and (3R, 4S) diastereomers. However, the yield of the desired (3S, 4S) diastereomer in the reduction step is low (15%) and the Raney nickel used for the reduction can cause racemization during the reduction step. Further, the separation step comprises silica gel chromatography of the crude reduction product, containing both (3S, 4S) and (3S, 4S) diastereomers, which represents a limitation to scale-up to preparing large lots of material. Then, in a 1987 reference, Jouin et al., EPO published application No. 210 896, ACHPA was prepared starting from Meldrum's Acid, a less-readily available starting material.
It was therefore an object of this invention to develop a method of synthesizing ACHPA which experienced minimal racemization and consequently little loss of optical activity. It was also an object to develop a process which could be scaled-up to allow production of optically-active ACHPA in large lots.